Novel sex-dependent differentially methylated regions are demethylated in adult male mouse livers

https://doi.org/10.1016/j.bbrc.2015.04.137Get rights and content

Highlights

  • We found novel Sex dependent diffeneretially methylated regions in mouse liver.

  • DNA methylation status in S-DMRs changed by testosterone dependent manner.

  • DNA methylation would contribute to sex dimorphic chromatin decondensation.

Abstract

In mammalian livers, sexual dimorphisms are observed in tissue-specific functions and diseases such as hepatocellular carcinoma. We identified sex-dependent differentially methylated regions (S-DMRs) which had been previously been characterized as growth hormone- STAT5 dependent. In this study, we performed genome-wide screening and identified ten additional hypomethylated S-DMR gene regions in male livers. Of these S-DMRs, Uggt2 and Sarnp were hypomethylated in both male and female livers compared to brain and embryonic stem (ES) cells. Similarly, Adam2, Uggt2, and Scp2 were hypomethylated in female embryonic germ (EG) cells and not in male EG cells, indicating that these S-DMRs are liver-specific male hypo-S-DMRs. Interestingly, the five S-DMRs were free from STAT5 chromatin immunoprecipitation (ChIP) signals, suggesting that S-DMRs are independent of the growth hormone-STAT5-pathway. Instead, the DNA methylation statuses of the S-DMRs of Adam2, Snx29, Uggt2, Sarnp, and Rnpc3 genes were under the control of testosterone. Importantly, the hypomethylated S-DMRs of the Adam2 and Snx29 regions showed chromatin decondensation. Epigenetic factors could be responsible for the sexual dimorphisms in DNA methylation status and chromatin structure, as the expression of Dnmt1, Dnmt3b, and Tet2 genes was lower in male mice compared to female mice and TET2 expression recovered following orchidectomy by testosterone treatment. In conclusion, we identified novel male-specific hypomethylated S-DMRs that contribute to chromatin decondensation in the liver. S-DMRs were tissue-specific and the hypomethylation is testosterone-dependent.

Introduction

In mammalian livers, sexual dimorphisms are observed in tissue-specific functions such as metabolism of sex steroids [1]. Sexual dimorphisms are more prevalent in diseases such as hepatocellular carcinoma (HCC), which is more common in men and postmenopausal women who exhibit high levels of serum testosterone [2]. Sexual dimorphism is also observed in animal models for HCC; male mice that express the hepatitis C virus (HCV) capsid protein (core protein) in the liver (male core Tg mice) preferentially develop HCC [3].

The sex differences in drug and sex steroid metabolisms result from sexual dimorphic expression of cytochrome P450 genes in the liver that are associated with differential DNA methylation of gene promoter regions [4]. Cyp2d9, a gene that encodes testosterone 16 alpha-hydroxylase, is highly expressed in livers of males and exhibits hypomethylation of its promoter region [4]. Such sex-dependent differentially methylated regions (S-DMRs) have been reported for several sex-dimorphic genes [5], suggesting involvement of DNA methylation in sexually dimorphic gene expression and stability.

Another crucial mechanism in hepatic sexual dimorphism is the growth hormone -STAT5 pathway (GH-STAT5 axis) [6]. The sexually differential patterns of serum GH [7] secreted from the pituitary cause differential activation of STAT5. Deficiency of Stat5b [6] or hypophysectomy [8] results in the loss of most sexual dimorphic gene expression, which can be recovered by GH injection. Sexually dimorphic GH secretion patterns are governed by sex steroids [9].

STAT5 binding sites are often associated with decondensed chromatin structures, indicated by their enrichment in DNase hypersensitive sites (DHS) [10]. Chromatin structure is governed by epigenetic mechanisms including DNA methylation and histone modification. DNA methylation is often associated with condensed chromatin [11]. DNA methylation patterns in several male-biased genes can be modified by female-type GH administration [5]. These data suggest that the GH-STAT5 axis has an impact on local chromatin structure through an epigenetic system including DNA methylation. In addition, neonatal androgenization affects the DNA of several promoters in adult mice including those ofCyp7b1 and Hnf6 genes [12].

DNA methylation profiles, which consist of numerous genome-wide, tissue-dependent, differentially methylated regions (T-DMRs), change during cell differentiation and development and in response to environmental factors [13], [14]. In this study, we investigated S-DMRs in the mouse liver and the effect of sex steroids on the DNA methylation profiles of adult mouse livers.

Section snippets

Animals and cells

C57BL/6N mice were obtained from Charles River (Yokohama, Japan). Orchidectomy (ORX) or ovariectomy (OVX) was performed in six male and female mice at 10 weeks of age; the mice were sacrificed 4 weeks later. Siliconized tubes (TP: 2 cm, EB: 0.5 cm) containing testosterone propionate (TP) (WAKO, Osaka, Japan) or estradiol benzoate (EB) (Sigma–Aldrich Japan K.K. Tokyo, Japan) were subcutaneously implanted into three ORX mice or three OVX mice, respectively and kept for 4 weeks. The effects of

Identification of male hypo-S-DMRs

We screened S-DMRs hypomethylated in male mouse liver by using D-REAM, a microarray-based genome-wide DNA methylation analysis method, and found S-DMRs to be less methylated in male livers (male hypo-S-DMRs) than in female livers [14]. Using D-REAM, we identified a male hypo-S-DMR at Cyp2d9 (Fig. S2), a locus previously shown to carry an identified S-DMR [4]. In addition, COBRA of the 10 identified S-DMRs indicated they were significantly hypomethylated (>10% difference in average DNA

Testosterone affects DNA methylation of S-DMRs

Genome-wide DNA methylation analysis allowed us to identify S-DMRs that were specifically hypomethylated in male mouse livers. Although some S-DMRs exhibited tissue-specific methylation patterns (i.e., less methylation in the liver than other tissues in both sexes), levels of DNA methylation at these S-DMRs were lower in the livers of male than in female mice. We also found that testosterone had an impact on DNA methylation at the identified S-DMRs in livers of adult male mice.

Previous studies

Availability of supporting data

The array data from this study have been submitted to ArrayExpress (http://www.ebi.ac.uk/microarray-as/ae/) under accession no. E-TABM-551 and no. E-TABM-789.

Conflict of interest

None.

Acknowledgments

The ES cell line MS12 was kindly provided by Dr. H. Suemori and Dr. T. Wakayama provided the ES cell lines mB6-1, fB6_1, and fB6-2. The mJ1 cell lines were purchased from the Cell Engineering Division of RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). BRC6(AES0010) and B6-6(AES0172) ES cell line used in this study were originally generated by M. Hirose and A. Ogura and were provided from the RIKEN Bioresource Center. We thank Dr. Atsuo Ogura for providing BRC6(AES0010) and B6-6 ES cell

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